Back to bases

July 06, 2012

I’m gearing up to blog in a big way about a huge blind spot in classical pharmacology: chronic drug accumulation in human tissues. Protein supremacist experimental approaches have nurtured in pharmacologists an unhealthy obsession with high-affinity “drug targets,” which shrouds, and in some cases distorts, decades-old insights by physiologists into the complex mode of action of therapeutic compounds that appear structurally simple.

To warm up, I’ll present a beautiful little paper by Hiruma & Kawakami (Folia Histochemica et Cytobiologica 2011; Vol. 49, No. 2, pp. 272–279), which resuscitates dormant observations about the effects of weak bases on living cells. The authors measured the response of several essential cellular components to high concentrations of the water-soluble weak base 4-aminopyridine in primary cell cultures derived from dissected mouse dorsal root ganglia.

As shown by light microscopy (Figure 1, above), 4 mM 4-aminopyridine caused many internal compartments (vacuoles) to appear, e.g., crater-like structures in panel B, especially at the 90-minute mark. Washing away the 4-aminopyridine after a brief (minutes to hours) treatment caused all those induced vacuoles to disappear gradually. However, long-term (hours to day) treatment with 4-aminopyridine induced irreversible “vacuolization,” which was toxic to cultured neurons and non-neurons alike.

Christian de Duve, proposed a theory called lysosomotropism to describe the cellular accumulation of weak bases. It invokes the Henderson-Hasselbach equation to explain the skewed subcellular distribution of weak bases, which accumulate in acidic compartments, even synaptic vesicles.

So what, you say? 4 mM of anything you can buy from Sigma would eventually kill cells “by a non-specific mechanism.” What’s happening to other dynamic, essential cellular components besides acidic compartments during a 4-aminopyridine overdose?

Well, the authors did a few controls (Figure 3, below):

First, they verified that the 4-aminopyridine-induced vacuoles observed under differential interference contrast (DIC) are indeed lysosomes by using a fluorescent lysosomal protein marker. Next they showed that the mitochondrial and actin filament networks are basically unaffected by an acute 1-hr treatment with 4-aminopyridine (4-AP).

Then they performed a bunch of before-and-after 4-AP treatment experiments with LysoTracker Green, which “stains acidic compartments,” according to the manufacturer. I’m not exactly sure what to make of these data, other than that vacuolization is probably being conflated with the induction of autophagy.

The specificity pièce de résistance is Figure 5 (below). The V-ATPase complex, responsible for acidifying organelles, is essential for the initial vacuole induction and the ensuing cascade of physiological effects:

To be honest, parts of the discussion suffer a tad from lost in translation. Throughout the paper, I got tripped up when the authors referred to organelles within vacuoles. I assume they were talking about autophagosomes or autophagolysosomes. The fact that these words don’t roll off my tongue and I’m a native English speaker attests to the potential for confusion.

The authors concluded the paper with the following model:

In addition, the contents of vacuoles are serous without proteins or amino acids and also without weak base. Thus, it is possible that vacuoles are formed by extrusion of H+ from acidic organelles along with water. Further studies are needed to prove this.”

I never quite understood what they meant by “serous vacuoles,” a phrase they repeated over and over. My interpretation is that they are seeing evidence of phospholipidosis. How weak base accumulation actually triggers phospholipidosis is a whole nother story…

I encourage whoever is interested to read the entire paper here. I’m hosting a discussion of it below. Who wants to break the ice?

 

 

 

 

 

 

 

 

 

  • Jeff Krise

    I enjoy the topic and believe that there is much to be understood regarding the physical interaction of amines with lysosomes and the contribution this plays on therapeutic activity and/or toxicity. The work presented in this manuscript helps shed light on this topic. However, I do have difficulty in reconciling the notion that the vacuoles are free of protein, amino acids and chloroquine though. It seems that the lack of fluorescence of CFDA-SE inside the vacuole is the primary evidence used to support the lack of protein and amino acids. I believe that it is possible that the vacuoles contain protein and the lack of CFDA-SE fluorescence could be artefactual. It its parent form dye is not fluorescent and is membrane permeable and amine reactive, thus labeling proteins. When the dye encounters intracellular esterases it becomes membrane-impermeable and fluorescent. I believe cellular esterases are associated with microsomes (surface of the ER) so it seems reasonable that the dye becomes fluorescent and membrane-impermeable in the cell cytosol. Being impermeable, the fluorescent molecule would not be able to diffuse into lysosomes/vacuoles in order to react with resident proteins even if they were abundant. Regarding the lack of chloroquine in the vacuole, this too could be artefactual. As I understand it, the lack of CQ in vacuoles was confirmed by the lack of CQ antibody staining. It seems that one would need to permeabilize the cells to get the CQ antibody into the cells, a process which would also release any sequestered small molecule such as CQ. I would enjoy hearing others opinions.

    Jeff Krise
    University of Kansas

    • http://twitter.com/eperlste Ethan Perlstein

      Thanks for the great review, Jeff!

      I agree that their claim that the induced vacuoles are protein-free is overstated. I was confused by their language use, referring to the induced vacuolar contents as “serous.” I wasn’t aware for the potential for an artifact with CFDA-SE, but your reasoning is sound. And I agree about the chloroquine Ab result being iffy as well.

      In your mind does is the vacuolization driven by osmotic swelling? And it seemed to me the inclusions subject to Brownian motion were autophagolysosomes.

      • Jeff

        The question about vacuolization and the osmotic driving force behind it is one that I have thought about quite a bit. I think that the osmotic pressure build up may trigger vacuolization but not as directly as may be assumed. I would reason that the lysosomal lipid bilayer, assuming fixed composition, would not be able to increase in size very dramatically under even extensive osmotic pressure but instead would rupture if the force were great enough (like red blood cells do). The finding that the lysosomes do not seem to burst but instead increase in diameter so dramatically suggests that lysosome fusion/fission processes that regulate lipid bilayer flux into and out of lysosomes must be implicated in vacuolization. It is possible that some of these trafficking steps involved could be influenced by osmotic pressure. I enjoy this site.

        Jeff

  • Teja Groemer

    Dear Ethan, I am really becoming a fan of your page. After my paper on accumulation, the resonance was mixed (see the discussion on schizophreniaforum.org) “Back to bases” really makes the point! Best, Teja

    • http://twitter.com/eperlste Ethan Perlstein

      Thanks, Teja! I read those posts on schizophreniaforum, too. Phillip Seeman is obviously a well-respected expert in pharmacology. I cite his 1972 paper (http://www.ncbi.nlm.nih.gov/pubmed/4565956) all the time.

      But I think he represents the protein-centric bias of classical pharmacology, which is interesting because in the late-60s and early-70s he was right in the thick of the debate over membrane vs protein effects of anesthetics. Whenever I talk to people about using yeast as a model for neuropharmacology, I experience the protein-centric bias 9 out of 10 times.

      Hopefully your Neuron paper will get people thinking beyond simplistic measurements of in vivo drug concentrations like serum or CSF levels, which obviously don’t report on subcellular accumulation, or seem to acknowledge the Chemistry 101 distinction between molarity and effective molarity.