Cell Physiology and Biophysics1

Cell forms the building block of life. They are highly specialized in various organs to carry out specific functions, but the structures present inside, namely the organelles are identical in all types of cells (Fig. 1.1). With the advent of recent techniques in the cell biology, it is now possible to separate organelles by ultracentrifugation.

Attached to the peripheral proteins on the exterior, mucopolysaccharides are present. The important constituent of this is sialic acid and its presence on the cell membrane gives negative electrical charge to the cell surface. These glycoproteins are also responsible for the antigenicity of the cell membrane.

The lipids and proteins on the cell membrane are in a dynamic state, in the sense, that they are in a fluid mobile state and can change their shape and position. In addition, there is also turnover of membrane lipids and proteins.

They are the structures present inside the cell. It includes nucleus, endoplasmic reticulum, Golgi apparatus, mitochondria, lysosomes, microsomes, microtubules and microfilaments. The detailed descriptions of these structures are given below.

Nucleus is surrounded by a double layered membrane with pores 70 nm size. These pores serve as transport channels for RNA to migrate to cytoplasm. Nucleus contains chromatin and one or more nucleoli. The nucleoli contain RNA, while, the chromatin has the DNA, which carries the genetic information. The structure of DNA shows two strands, which are folded into a double helical structure called chromosomes. The number of chromosomes present in the nucleus of human being is 46, which includes 22 pairs of autosomes and one pair of sex chromosomes.

It is one of the constituents of microsomes. It is of two types namely, smooth endoplasmic reticulum (SER) and rough endoplasmic reticulum (RER). The rough endoplasmic reticulum consists of flat vesicles which are interconnected to form a network of channels throughout the cell. The surface of the RER contains ribosomes on which the proteins are synthesized.

It consists of stacks of flat vesicles and is closely associated with RER. Golgi apparatus receives the synthesized protein and modifies it by concentration, processing and packaging. The concentration of the synthesized molecule occurs by the removal of water, while processing involves cleavage of the precursor molecule and addition of carbohydrate moiety. The packaging of the synthesized molecule forms the secretory vesicle. The secretory vesicle is transported to the surface of the cell guided by the microtubules and released from the cell by exocytosis.

It is oval shaped and has double layered cell membrane. The inner membrane has been thrown into folds called cristae which increase the surface area. The cristae includes the matrix (Fig. 1.3). The respiratory enzymes are present in the cristae while the matrix contains the nuclear material DNA and RNA. The RNA is involved in the synthesis of enzymes of oxidative phosphorylation, while the DNA facilitates mitochondrial replication. The mitochondria is called the power house of the cell, as the energy rich compound ATP is synthesized from them. The number of mitochondria in a cell varies in relation to its metabolic activity. The cells that are concerned with the secretion and those that are metabolically active, such as liver cells, will have more number of mitochondria.

They are the vesicles containing hydrolytic enzymes. They are formed from endoplasmic reticulum and Golgi apparatus. The main function of lysosomes is in the digestion of the engulfed material. The substances that are engulfed into the cell by pinocytosis or phagocytosis, fuse with the lysosomes and form the digestive vacuole. The lysosomes which are not attached to the phagocytic vacuole are known as primary lysosomes and those which are attached are called secondary lysosomes. The acid hydrolases in lysosomes digest the engulfed material and the indigestible ones are in the residual body. It is released from the cell by exocytosis. The lysosomes also can digest the breakdown products of cell's own organelles. In certain pathological conditions such as gout, rheumatoid arthritis, the release of lysosomal enzymes is considered to be the causative factor in the pathogenesis of the disease.

It is one of the constituents of microsomes and can be obtained by ultracentrifugation of the cell organelles. The peroxisomes are more in number in liver and kidney. They contain an enzyme called catalase which causes breakdown (detoxification) of hydrogen peroxide. They are also involved in theβ oxidation of lipids and thereby regulate their level in the plasma.

The cell contains fibers which include microtubules, intermediate filaments and microfilaments. These are known as cytoskeletal structures. They give structural stability to the cell and produce cell movement.

The microtubules give shape to the cell. They facilitate the transport of secretory vesicles and organelles to the cell surface. The microtubules are in a dynamic state as they can assemble and disintegrate in various conditions.

Microfilaments are made of actin and their interaction with myosin I cause cell movement. Most of the cells contain actin at the cell surface. The interaction of actin with myosin II gives muscle contraction.

Cilia and flagella are the cell processes surrounded by the extensions of plasma membrane. The intercellular structure consists of microtubules. There is an outer 9 units of microtubules and an inner pair present in the center. The movement of cilia or flagella is caused by the sliding of microtubules.

Cell adhesion molecules(CAMs) are proteins, which facilitate attachment of the cell to basal lamina and between cells. Some of these molecules also project into the cell and are attached to the cytoskeleton. CAMs are broadly divided into integrins, IgG superfamily of immunoglobulins, cadherins and selectins.

The cells are interconnected mainly in three ways namely tight junction, gap junction and desmosome (Fig. 1.4).

It provides gaps or channels from one cell to another and allows the passage of small molecules and ions.5

In smooth muscles and cardiac muscle, the electrical excitation spreads to adjacent cells by the movement of ions through the gap junctions.

It is a connection between two adjacent cells at the apical surface. The fusion of the lateral borders at the apical surface forms a barrier against the entry of substances. This kind of intercellular connection is seen in the intestinal epithelial cells, renal tubular cells and choroid plexus.

Two adjacent cells are held together by the electron dense fibrous substance in the extracellular space. There are intermediate filaments projecting from each cell, at the point of cell adhesion. The usual width of this connection is 20 nm. This type of connection between two cells helps to overcome mechanical stretching that occurs in the tissues like muscle and skin.

The plasma membrane forms the boundary between the cell interior and exterior. The membrane shows transport of substances across the membrane in both ways.

The uptake of molecules into the cell occurs by a process called endocytosis. The molecule at first fuses with the cell membrane and invaginates. The invagination is separated from the cell membrane and becomes a vesicle.

They are also other proteins namely clathrin, actin and myocin present beneath the receptors, which help the pinocytic vesicle to pinch off from the cell membrane. The process of endocytosis requires the supply of ATP as it is an active process.

Exocytosis refers to the release of substances from the cell. It could be the proteins synthesized by the RER or the cell organelles that are disintegrated. The synthesized proteins form the secretory vesicle and are guided by microtubules to the cell surface. The membrane of the vesicle and plasma membrane fuse and the contents of the vesicle are extruded out (Fig. 1.6). This process requires Ca⁺⁺ entry into the cell and supply of energy.

Transcytosis also can be observed in the transport across the membrane. It refers to the movement of macromolecules (protein hormones) from one side of the cell to the other side unchanged. This kind of transport is seen in endothelia.

When there is a concentration gradient across the membrane, diffusion of water and solutes occur. The diffusion will be from a region of higher concentration to a region of lower concentration (Fig. 1.7).

Diffusion of solutes across the membrane follows Fick's law. It states that the rate of 7diffusion (J) is directly related to the cross sectional area (A) through which diffusion takes place and concentration gradient (C/X)

D = diffusion coefficient and– sign denotes the direction.

Water molecules move down along the concentration gradient through a semipermeable membrane and this process is called osmosis (Fig. 1.8). It is the movement of solvent into a region of greater concentration of solute to which the membrane is not permeable. Examples of water diffusion by osmosis are seen in renal tubules, intestinal mucosa, between cells and exterior, between capillaries and interstitial fluid. The process of osmosis creates hydrostatic pressure in the region to which the solvent diffuses. The movement of solvent can be prevented by applying pressure over the region of higher solute concentration. This pressure is known as osmotic pressure. It depends on the number of solute particles and in an ideal solution, the osmotic pressure (p) is related to:

n = number of solute particles

R = gas constant

T = absolute temperature

V = Volume

Whenever solvents diffuse, they tend to drag some solute with it. This is called solvent drag, and can be observed in capillaries and renal tubules.

A substance that cannot diffuse through the pores or leaky channels in the membrane can be transported by a carrier across the membrane.8

Lipid soluble substances and respiratory gases can easily diffuse through the membrane. There are also substances of low molecular size, which can pass through the leaky channels. The solutes that cannot pass through these pores depend on the carrier proteins to transport them. If the transport occurs along the concentration gradient, it becomes a facilitated diffusion. It is a downhill transport, because, the transport does not require energy.

Facilitated diffusion is determined by:

The carrier protein shows:

There are instances where two solutes are transported together by the carrier and such transport is called symport or cotransport. Examples of this transport are glucose transporter (SGLUTI) which transports both glucose and Na⁺ from the lumen of intestine to the mucosal cell (Fig. 1.10). Amino acids are also transported as symport with Na⁺ in the intestine. Similar cotransport also exists in the renal tubules.

Active transport of solutes occurs from a region of lower concentration to a region of higher concentration. This requires a carrier protein and the supply of energy, as it is an uphill transport (Fig. 1.11). Examples of active transport include Na⁺ pump, secretion of H⁺ from the parietal cells of gastric glands, iodide transport in the thyroid gland acinar cells, etc.

Active transport of Na⁺ by the Na⁺-K⁺ ATPase carrier across the basolateral border of the intestinal mucosal cells and renal tubules, results in entry of glucose into the cell. As said above, glucose and Na⁺ are cotransported from the lumen into the cell. The active transport of Na⁺ in the basolateral surface provides the chemical gradient for further movement of Na⁺ and glucose into the cell. Glucose transport into the cell forms the secondary active transport, as it depends on the active transport of Na⁺ at the basolateral surface (Fig. 1.12).

The solutes, when exchanged in the opposite direction by the carrier protein, form the counter transport or antiport (Fig. 1.13). Examples are:

Donnan and Gibbs have shown that the sum of anions and cations on either side of the cell membrane are equal, eventhough their distribution across the membrane varies due to the selective permeability of the membrane. It is known that unequal distribution of ions gives electrical potential. The cations and anions on either side of the membrane are distributed in such a way that in each compartment their total number will be equal. This is called Donnan's electrochemical equilibrium.

The unequal distribution of ions across the cell membrane, selective permeability of the cell membrane for K⁺ and the electrogenic Na⁺– K⁺ pump creates potential difference between inside and outside of the cell membrane. In excitable tissues namely nerve and muscle this electrical potential is significant for excitation of the cell. In these tissues the electrical potential does not alter the Donnan's ionic equilibrium as only a small number of ions are involved in the genesis of membrane potential.

Communication from one cell to another usually takes place by the chemical messengers. These substances can be proteins, amino acids or lipids. Chemical messengers enter the cell through gap junctions, paracrine, endocrine and neural communications. There is also cell's own secretion that can act on itself which forms the autocrine secretion.

The examples of second messengers are:

Second messengers are formed through a nucleotide regulatory protein called G protein. When a first messenger (ligand) binds to the receptor on the cell membrane, the ligand receptor (Gs) complex converts GTP to GDP and activates adenylyl cyclase, if cyclic AMP is formed as second messenger. The binding of the ligand with the Gi inhibits the adenylyl cyclase activity and prevents the formation of cyclic AMP. Cyclic AMP activates protein kinase A, which phosphorylates proteins, leading to the physiological effects (Fig. 1.14). The cyclic AMP is inactivated by the enzyme phosphodiesterase, which converts 3’ 5’ AMP to 5’ AMP and it is inactive. If this enzyme is inhibited, the activity of cyclic AMP can be prolonged. Substances like caffeine, theophylline show such effect when administered.

Similar to cyclic AMP, there is another nucleotide known as guanosine mono phosphate (cyclic GMP), which functions as the second messenger. Atrial natriuretic peptide (ANP) and nitric oxide (NO) mediate their effects through cyclic GMP. In the retina, during excitation of rods, GMP is converted to 5 GMP which is responsible for the photochemical changes.

In the formation of IP₃ and DAG as second messengers, the ligand receptor (Gs) complex converts GTP to GDP and activates phospholipase enzyme. This facilitates the formation of IP₃ and DAG from the phosphorylation of phosphatidyl inositol released from cell membrane.

The binding of ligand with the receptor on the cell surface, opens up the Ca⁺⁺ channels, facilitating its entry into the cell. Inside the cell, the calcium binds to a protein calmodulin and this complex causes activation or inactivation of enzymes. Through these changes, the physiological effects of the ligand can be observed (Fig. 1.16).

Hormones such as T₃ (tri iodo thyronine) has receptors in the nucleus. The hormone after binding to the receptor in the nucleus, stimulates transcription of DNA in the chromatin. This leads to mRNA formation, which leaves nucleus and enters the endoplasmic reticulum. Here, new proteins are synthesized by translation. The synthesized protein can be an enzyme, secretory protein, structural protein, or receptor protein. The biological effects of the ligand are mediated through these new proteins (Fig. 1.17).

As described earlier, the cell membrane has structural proteins, which project on the outer and inner surface of the membrane. It is the peripheral proteins present on the outer surface of the membrane which function as receptors for ligands and neurotransmitters. These receptors are not static structures, as they are dynamic and mobile on the cell membrane. Their number is also not constant, as there is addition and removal to their numbers during the process of regulation.

Ion channels are formed in the cell membrane by the integral proteins. These channels are guarded by gates, which open and close, regulating the movement of ions. The gates are regulated either by voltage or chemical ligands. Accordingly, the channels are called voltage gated and ligand gated channels respectively.

Ion channels can be studied by patch clamp technique (Fig. 1.18). A microelectrode is kept on the surface of the membrane and a high resistance seal is applied around the tip of the microelectrode. By a suction force, a patch of cell membrane containing a few ion channels is taken inside the seal. The activity of the ion channels can be studied in situ or the small part of the membrane is removed and kept in solutions of known ion concentration to study the channel activity.

It is a tetramer with four units. Each unit crosses the membrane six times. The pores with 0.5 nm diameter is present surrounding the subunits. The channel is guarded by gates, which are present in both inner and outer sides of the membrane. On the ECF side, there is activation gate. On the inner side of the membrane, there is an inactivation gate. For sodium to enter inside, both the gates should be in the open state (Fig. 1.19). The sodium channels are blocked by poisons like tetrodotoxin and saxitoxin.

The structure of the potassium channel is similar to the sodium channel and they are the voltage gated channels. There is another type of K⁺ channel which has only two subunits and crosses the membrane twice. These are called inward rectifier potassium channels which allow K⁺ entry and not its exit. Unlike Na⁺ channel, K⁺ channel (voltaged gated channel) has only one gate and it is present on the inside of the membrane. There is no inactivation gate for the potassium channel. The gate should open for K⁺ movement (Fig. 1.20). The voltage gated potassium channels can be blocked by chemicals like TEA (tetra ethyl ammonium) and 4 - amino pyridine. Ligand gated potassium channels also exist and activated by:

Calcium and chloride channels are similar to sodium channel in their structure. The ligand gated Ca⁺⁺ channel is activated by hormones and neurotransmitters. The voltage gated Ca⁺⁺ channel are of three types namely: